Cost-Efficient Autotrophic High-Cell-Density Cultivation of Cupriavidus necator Enabled by Model-Based Gas Supply.

Cultivating hydrogen-oxidizing bacteria (HOB), such as Cupriavidus necator, using H 2 ${{\rm{H}}}_{2}$ , CO 2 ${\text{CO}}_{2}$ , and O 2 ${{\rm{O}}}_{2}$ offers a promising route for CO 2 ${\text{CO}}_{2}$ valorization into chemicals and materials. To enhance cultivation efficiency in a lab-scale gas fermenter lacking a gas recycling system, an automated gas supply strategy based on real-time CO 2 ${\text{CO}}_{2}$ and O 2 ${{\rm{O}}}_{2}$ monitoring was developed. Fine-tuning gas delivery is essential to ensure an adequate supply for cellular growth while minimizing excess gas, particularly H 2 ${{\rm{H}}}_{2}$ , that leaves the bioreactor unused, to improve process economics. In the absence of ATEX-compliant H 2 ${{\rm{H}}}_{2}$ sensors, a soft sensor was implemented to estimate dissolved H 2 ${{\rm{H}}}_{2}$ concentrations from O 2 ${{\rm{O}}}_{2}$ uptake rates and growth phase identification. Total gas flow was controlled according to the O 2 ${{\rm{O}}}_{2}$ requirements of the cells. This strategy reduced overall gas and H 2 ${{\rm{H}}}_{2}$ consumption by 67%. In addition, a high-cell-density medium was formulated by integrating published recipes with Inductively Coupled Plasma Optical Emission Spectroscopy and nutrient inhibition testing. The optimized medium increased biomass yield from 15 g/L to 53 g/L, with 75% of the dry weight consisting of the bioplastic poly(3-hydroxybutyrate), without requiring nutrient addition or pH control. Together, these strategies improve the scalability, efficiency, and sustainability of CO 2 ${\text{CO}}_{2}$ -based cultivation of hydrogen-oxidizing bacteria.